Highly stable oxidative coupling dye for spectrophotometric determination of analytes

ABSTRACT

A dye couple compound is provided for use in a test device containing a reagent system for detecting the presence or quantity of an analyte in a sample. The reagent system comprises one or more enzymes which, in the presence of the analyte, produce an oxidizing agent in quantities indicative of the quantity of analyte in the sample. The compound of choice is meta[3-methyl 2-benzothiazolinone hydrozone]N-sulfonyl benzenesulfonate monosodium.

TECHNICAL FIELD

The present invention relates to a test device and method for thecolorimetric determination of chemical and biochemical components(analytes) in aqueous fluids, such as whole blood, and, moreparticularly, to a dye couple for use in such device and method.

BACKGROUND ART

The quantification of chemical and biochemical components in coloredaqueous fluids, in particular, colored biological fluids such as wholeblood and urine and biological fluid derivatives such as serum andplasma, is of ever-increasing importance. Important applications existin medical diagnosis and treatment and in the quantification of exposureto therapeutic drugs, intoxicants, hazardous chemicals, and the like. Insome instances, the amounts of materials being determined are either sominuscule--in the range of a milligram or less per deciliter--or sodifficult to precisely determine that the apparatus employed iscomplicated and useful only to skilled laboratory personnel. In thiscase, the results are generally not available for some hours or daysafter sampling. In other instances, there is often an emphasis on theability of lay operators to perform the test routinely, quickly, andreproducibly outside a laboratory setting with rapid or immediateinformation display.

One common medical test is the measurement of blood glucose levels ofdiabetics. Current teaching counsels diabetic patients to measure theirblood glucose level from two to seven times a day, depending on thenature and severity of their particular cases. Based on the observedpattern in the measured glucose levels, the patient and physiciantogether make adjustments in diet, exercise, and insulin intake tobetter manage the disease. Clearly, this information should be availableto the patient immediately.

Many blood glucose test methods and test articles are known in the art;these all suffer from a variety of limitations. A great improvement isdisclosed and claimed in U.S. Pat. Nos. 4,935,346, 5,049,487, 5,059,394and 5,179,005 by R. Phillips et al. and as assigned to the same assigneeas the present application.

The method disclosed and claimed in these patents involves taking areflectance reading from one surface of an inert porous matriximpregnated with a reagent that will interact with the analyte toproduce a light-absorbing reaction product when the fluid being analyzedis applied to another surface and migrates through the matrix to thesurface being read. The reagent includes glucose oxidase, an enzymewhich consumes glucose in the sample to produce hydrogen peroxide which,in the presence of another enzyme, horseradish peroxidase, oxidizes adye couple comprising 3-methyl-2-benzothiazolinone hydrazonehydrochloride (MBTH) and 3-dimethylaminobenzoic acid (DMAB) to yield ablue dye. Reflectance measurements are then made at two separatewavelengths. The concentration of the glucose in blood is determinedbased on the intensity of the dye color with the aid of a LEDspectrophotometer.

In my commonly assigned U.S. Ser. No. 245,940, filed May 19, 1994, nowU.S. Pat. No. 5,453,360, (LFS 30), there is disclosed a dye couplecomprising 3-methyl-2-benzothiazolinone hydrazone in free form or inacid form (MBTH) and 8-anilino-1-napthalenesulfonate, in acid or saltform (ANS) to be used in place of the MBTH-DMAB dye couple as describedabove. The MBTH-ANS dye couple is less soluble upon oxidation and,hence, provides a more stable endpoint, with minimal dye fading, ascompared to the oxidized MBTH-DMAB dye couple.

While these prior systems have been effectively employed to produceuseful test devices for the determination of the presence or quantity ofglucose, several drawbacks have been noted. The test devices in whichsuch dye couples are employed are designed for both home use and forprofessional use and as such are sold by the manufacturers anddistributors with the expectations that they will remain in theinventory of the user for a substantial period of time and must, ofcourse, remain effective over this period of time. This need for asubstantial shelf life has caused difficulties in the formulation ofproducts employing MBTH as one of the components of a dye couple.

Firstly, it has been found that the stability of MBTH decreases withincreasing temperature and alkalinity. The acid free form of MBTH isvery labile and tends to sublime away. In an attempt to counter this, apreferred form is the acid hydrate of MBTH e.g.,3-methyl-2-benzothiazolinone hydrazone hydrochloride. Unfortunately,this hydrate is itself instable upon increasing temperature and readilydissociates into acid free MBTH and HCl upon heating. In additional tohaving low stability at high pH, the efficiency of MBTH to oxidativelyreact with its coupling partner greatly decreases with increasingalkalinities so that at high pH essentially little or no color isproduced from the dye couple.

In view of these relationships, in practice MBTH must be used in largeexcesses and at low pH to minimize the effects of instability andinefficiency. Ideally, a pH of below 2.0 would be preferred from thepoint of view of low sublimation and high efficiency of the compound.Unfortunately, for the systems being considered herein such an ideal lowpH cannot be employed. As described above, the reagent systems employeddepend upon enzymes to act on the substrate analyte and generateoxidation agents in quantities indicative of the quantities of theanalyte present in the sample being tested. The low pH, which would beideal with respect to the MBTH reagent, is entirely unsuitable forenzymes such as, for example, glucose oxidase and horseradishperoxidase. At such low pHs many of such commercially available enzymeshave little or no activity. Accordingly, the art has been forced tocomprise and chose a moderate pH e.g., 4, and a great excess of reagentsto insure efficiency of their test devices for the required shelf life.

SUMMARY OF THE INVENTION

In accordance with the teachings of this invention, a highly stablecomponent of a dye couple is provided in a test device containingenzymes. The component, in contrast to those employed in prior testdevices, is capable of efficient oxidative coupling with a wide varietyof coupling partners at the relatively high pH conditions compatiblewith high enzyme efficiency.

Specifically, the dye couple compound of this invention is to be used ina test device containing a reagent system for detecting the presence orquantity of an analyte in a sample wherein the reagent system comprisesone or more enzymes which, in the presence of the analyte, produce anoxidizing agent in quantities indicative of the quantity of the analytein the sample. In accordance with the teachings herein, the reagentsystem further comprises a dye couple capable of forming a chromophoreupon being oxidized by the produced oxidizing agent; the dye couplecomprises the compound: ##STR1## wherein R is selected from the groupconsisting of alkyl, substituted alkyl, aryl, substituted aryl,heterocyclic, quaternary amine or organic acid moieties and Y isselected from the group consisting of NO₂, SO₃ --, H, halide, alkyl orSiZ₃ wherein Z is either alkyl or aryl. Preferably, Y is H. In apreferred embodiment R is: ##STR2## wherein any of R₁, R₂ and R₃ areindependently selected from the group consisting of H, alkyl, aryl,silyl, halide, hydroxide, mercaptide, alkoxide, thioalkoxide, amine,sulfonate or carboxylate; and X is selected from the group consisting ofamine, sulfonate or carboxylate. In a specific embodiment, the testdevice is provided to determine the presence or quantity of analytessuch as glucose, cholesterol, alcohol, uric acid, formaldehyde orglycerol-3-phosphate, all commonly measured blood analytes. In suchcases, the enzyme system will comprise enzymes selected from the groupconsisting of glucose oxidase, cholesterol oxidase, alcohol oxidase,uricase, aldehyde oxidase, and glycerophosphate oxidase; together withperoxidase or inorganic complex which has peroxidase-like activity;e.g., hematin, hemin and tetrakis[sulphophenyl]porphyrin manganese. Aperoxidase of choice is horseradish peroxidase.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a perspective view of one embodiment of a test devicecontaining a reaction pad to which the liquid sample being analyzed isapplied; and

FIG. 2 is a perspective view of a second embodiment of the employment ofthe test device of FIG. 1.

DETAILED DISCUSSION OF THE INVENTION

As described above, the invention involves an improved dye couplecompound for use in a test device for determining the presence orquantity of an analyte in a liquid sample. Referring to FIG. 1, in apreferred embodiment of this invention, the test device comprises aporous matrix 10 having incorporated therein a chemical reagent systemand being adhered to a support 12. An aperture 16 is provided throughthe support whereby a liquid sample may be applied to a sample receivingsurface 17 of the matrix 10. The chemical system is provided to reactwith any analyte present in the liquid sample and result in having atest surface 19 of the matrix manifest light reflectance propertiesindicative of the quantity of analyte present in the liquid sample. Thetest surface may be read with naked eye, but preferably is read by useof a spectra photo matrix device. Elements of such devices are shownschematically in FIG. 1 and comprise a light source 18 such as a lightemitting diode for directing preferably uniform wavelength light ontothe test surface 19. Additionally provided is light detector 20 fordetecting reflected light from surface 19 and producing a signal 22indicative of the quantity of detected light, which signal may beprocessed by, for example, a microprocessor incorporated into thereading apparatus to calculate the quantity of analyte in the sample.

Systems such as the one described above are now known in the art and arewell described in U.S. Pat. Nos. 4,935,346; 5,049,487; 5,059,394; and5,179,005. Such systems contemplate that these test devices and will beinserted into a reading apparatus and then the sample, e.g., blood, willbe applied to the sample receiving surface 17. FIG. 2 represents analternative to this, wherein blood is first applied to the samplereceiving surface 17 and only then is testing surface 19 presented tothe apparatus for reading. In all other respects, the numbered elementsin FIG. 2 are identical to those of FIG. 1.

The reflectance properties of the testing surface varies with theanalyte quantity in the sample by the operation of a series of chemicalreactions between the analyte in the liquid sample and the chemicalreagents present in the porous matrix. In particular, the matrixincludes one or more enzymes which, together with theanalyte-substitute, results in the production of hydrogen peroxide orother strong oxidizing agents. A dye couple is included in the matrix;i.e., two compounds which are capable of being oxidized to form achromophore which absorbs light at specific wave lengths in proportionto the quantity of chromophore present. The oxidizing agent formed bythe enzyme catalyzed reaction then reacts with the dye sample to producethe chromophore.

The choice of enzymes, the resulting oxidizing agent and the choice ofdye couple vary widely in the art, and are, to a great measure, afunction of which analyte is being determined. For example, in the caseof determination of cholesterol as in a blood sample, an oxidase enzymesuch as cholesterol oxidase may be employed. Similarly, methanol orethanol determination may employ alcohol oxidase; formaldehydedeterminations may employ aldehyde oxidase; or glycerol-3-phosphatedeterminations may employ glycerophosphate oxidase. The hydrogenperoxide product of these enzyme catalyzed reactions may be furthermodified by a subsequently enzyme catalyzed reaction to produce anactive oxidizing agent for reacting with the dye couple to form thechromophore. Thus, for example, the reaction of hydrogen peroxide toform an active oxidizing reagent may be catalyzed by the enzymehorseradish peroxidase.

Accordingly, while it will be understood that the teachings of thisinvention are widely applicable, for the purpose of the followingdiscussion, the analyte will be exemplified by glucose in a liquidsample of whole blood. The preferred chemical system will then beexemplified by the enzyme glucose oxidase which acts on the glucosesubstrate to form hydrogen peroxide. Hydrogen peroxide, in turn, isconverted into active oxidizing reagent by the reaction of anotherenzyme, horseradish peroxidase.

Heretofore, the dye couple widely employed in a diagnostic test forglucose of the kind described above was the combination of3-methyl-2-benzothiazolinone hydrazone, hydrochloride hydrate (MBTHhydrochloride hydrate) (Formula I) together with dimethylamino benzene(Formula II). These compounds undergo the following oxidization reactionto form a blue colored chromophore (Formula III): ##STR3##

As described above, this system suffers from several drawbacks. TheMBTH, even in the hydrochloride hydrate form, is relatively unstableunder the action of heat and alkalinity. Furthermore, the above reactionis most efficient under highly acidic conditions; e.g., pH of 2 or less.Unfortunately, at these conditions, the enzymes employed in the testdevices e.g., glucose oxidase and horseradish peroxidase, have little orno activity. Accordingly, commercial practice has dictated that in orderto get a relatively stable system, an optimum pH is employed; e.g.,about 4, and large quantities of both enzymes and the dye couple areused to make up for the decreased activity of the enzymes and thereduced efficiency of the oxidization of the coupling reaction.

In accordance with this invention, it has now been discovered thatmodified forms of MBTH may be provided which overcome the stabilityproblem heretofore encountered and moreover are efficiently reactive inan environment more conducive to the activity of the enzymes employed inthe test devices contemplated herein; e.g., at pH values ranging fromabout 4 to about 7. Certain preferred derivatives have, moreover, beenfound to be highly reactive with the desirable coupling partners thearomatic amines. The derivatives of the invention have the generalstructure set out in Formula IV, below: ##STR4## wherein R is selectedfrom the group consisting of alkyl, substituted alkyl, aryl, substitutedaryl, heterocyclic, quaternary amine or organic acid moieties and Y isselected from the group consisting of NO₂, SO₃ --,H, halide, alkyl orSiZ₃, wherein Z is either alkyl or aryl. Preferably, Y is H. In apreferred embodiment R is: ##STR5## wherein any of R₁, R₂, R₃, and R₄are independently selected from the group consisting of H, alkyl, aryl,silyl, halide, hydroxide, mercaptide, alkoxide, thioalkoxide, amine,sulfonate or carboxylate; and X is selected from the group consisting ofamine, sulfonate or carboxylate.

The MBTH derivatives of this invention can undergo an oxidative reactionwith a wide range of dye couple partners such as aromatic amines,phenols, and substituted phenols. Moreover, such reactions can proceedefficiently at room temperature and at pHs which may vary from 4 to 11.In the preferred form of the derivatives of this invention, theoxidation reaction is optimal at pHs of from about 4 to about 7 and,hence, is particularly useful in conjunction with the amine dye couplepartners of interest in diagnostic chemistry such as 3-dimethlyaminobenzoic acid and 8-anilino-1-naphthalenesulfonates.

Unlike the MBTH, either in the acid free or in acid hydrate form, thesederivatives are remarkably stable even when heated at 100° C. for asmuch as 16 hours. Moreover, at the conditions of the oxidizationreaction, the peroxide catalyzing enzymes, such as horseradish peroxide,are especially effective in turning over the oxidization couplingreaction.

EXAMPLE 1 Synthesis of the MBTH Derivative Synthesis of meta [3-methyl2-benzothiazolinone hydrazone]N-sulfonyl benzenesulfonate monosodium,[2] Synthesis Scheme ##STR6## Material

3-methyl 2-benzothiazolinone hydrazone hydrochloride (MBTH.HCl), NaI,tetrabutylammonium hydroxide, methylene chloride andn-methyl-2-pyrrolidone were purchased from the Aldrich Company ofMilwaukee, Wis. and used without purification. Triethylamine wasobtained from Baker Chemicals and distributed by Baxter Company ofPhillipsburg, N.J. The 1,3 disulfonylchloride benzene was purchased fromFluka Chemicals of Ronkonkoma, N.Y. or Lancaster Chemicals of Windham,N.H.

Synthesis of [1]

A 4 gm sample of MBTH.HCl was charged into a 150 ml Erlenmeyer flaskequipped with a magnetic stirring bar, and 50 ml ofn-methyl-2-pyrrolidone and 5 ml of triethylamine were added. The flaskwas capped with a rubber septum and placed on a magnetic stirring hotplate. The mixture was heated to 60°-70° C. while stirring vigorouslyfor 0.5 hr., yielding a yellow slurry. The flask was placed into an icebath to cool.

A 5 gm sample of 1,3 disulfonylchloride benzene was added to a 250 mlErlenmeyer flask equipped with a magnetic stirring bar. The flask waslowered into an ice bath, and 20 ml of n-methyl-2-pyrrolidone was added.The mixture was stirred until all the solid was dissolved. (ca. 15 min.)The MBTH free-base slurry, which was obtained previously, was decantedinto the solution. The resulting light-yellow mixture was allowed toreact at an ice-bath temperature for 1.5 hr. After which time, thereaction was quenched with 10 ml of 2N HCl, and it was stirred for anadditional 30 min. at room temperature. 50 mg of 12 mesh Norti(activated carbon pellets) was introduced to the solution, affording alight yellow solution after 10 min of stirring. It was then filteredthrough a fine graded frit with the aid of an aspirator. A yellow tolight brown smooth solution was obtained. 300 ml of 2N HCl was added tothe stirring yellow solution, resulting in precipitation of an off-whitepowder. The solid was collected via vacuum filtration and the productwas washed 3 times with 25 ml deionized water. Upon drying at 110° C. invacuum for 2 hr. 5.6 gm of the off-white product was obtained. Theproduct was analyzed with ¹ H NMR and HPLC to be 97% pure.

Compound [1] is not very soluble in most of the common organic solventsand water. It is, however, soluble in basic solution and polar solvents,such as DMSO, NMP and DMF.

Synthesis of [2]

A 2.0 gm sample of the crude [1] was suspended in 50 ml methylenechloride. 4 ml of 1M tetrabutylammonium hydroxide was added slowly, in acourse of 2 min, to the stirring suspension; affording a light yellowsolution. The solution was washed with 10 ml deionized water and driedover anhydrous sodium sulfate. The sulfate was removed via gravityfiltration, and the resulting mixture was evaporated to dryness with arotary-evaporator. A thick yellow oil was collected. The oil was takenup with 125 ml acetone, and 10 ml of 20% NaI in acetone was added in thecourse of 5 min. A white precipitant was apparent. The mixture wasallowed to react for an additional 20 min., and the precipitant wascollected via vacuum filtration with a fine-grade frit. The resultingoff-white solid was washed 3 times with 20 ml acetone. Upon drying at110° C. for 45 min, 1.3 grm (65%) of the desired product was obtained.

Compound [2] is very soluble in water and in a water-alcohol mixture.The solid is stable in air and light, but its solution decomposes slowlyto a light yellow hazy mixture when exposed to light for a prolongedperiod of time.

EXAMPLE 2 Preparation of a Test Device

A strip of polymer membrane (reaction matrix) is submerged into theaqueous dip in Table 1 until saturated. It is removed from the dip andthe excess reagent is squeegeed off with a glass rod. The strip is thenhung inside an air circulating over at 56° C. for about 5-10 minutes todry, after which time the strip is removed and dipped into the organicdip described in Table 1 until saturated. Again, it is then dried as inthe previous step. The resulting strip is fashioned into a desired shapefor testing.

                  TABLE 1                                                         ______________________________________                                        Formulation of Reagents                                                       Aqueous Dip                                                                   (Adjust pH to 4.25                                                            with NaOH)         Organic Dip                                                ______________________________________                                        Water        20 ml     Water         3 ml                                     Citric acid  420 mg    Denatured     7 ml                                                            Alcohol                                                Ethylene diamine                                                                           16.7 mg   Meta[3-methyl 2-                                                                            10-                                      tetraacetic acid       benzothiazolinone                                                                           60                                       (EDTA)                 hydrazone     mg                                                              N-sulfonyl                                                                    benzenesulfonate                                                              monosodium [2]                                         Gantrez S95  90 mg     ANS           10-                                      (available from                      100-                                     GAF, New York,                       mg                                       New York                                                                      Crotein SPA  250 mg                                                           (available from                                                               Croda Co., New                                                                York, New York                                                                Glucose oxidase                                                                            20,500                                                                        units                                                            Horseradish  16,200                                                           peroxidase   units                                                            ______________________________________                                    

EXAMPLE 3 Determination of Glucose

A glucose containing blood sample is applied onto the surface of thereagent impregnated strip. The sample is immediately absorbed into thematrix and a blue color is apparent. The intensity of color increaseswith time and is proportional to the concentration of analyte. Based onthe color intensity, the glucose concentration is determined bycomparing with a standard calibration curve.

Similarly, an aqueous solution of hydrogen peroxide (organic peroxides,ferric and quinone) also produces the desired blue color on the reagentimpregnated strip. The concentration of the analyte may be determined bythe same means as above.

The invention now being fully described, it will be apparent to one ofordinary skill in the art that any modifications and changes may be madethereto without departing from the spirit and scope of the presentinvention.

What is claimed is:
 1. In a test device containing a reagent system fordetermining the presence or quantity of an analyte in a sample whereinsaid reagent system comprises enzymes to produce an oxidizing agent inquantities indicative of quantities of said analyte in said sample; theimprovement wherein:said reagent system comprises a dye couple forming achromophore upon being oxidized by said oxidizing agent at pH less thanabout 11, said dye couple comprising a compound having the formula:##STR7## wherein R is selected from the group consisting of alkyl,substituted alkyl, aryl, substituted aryl, heterocyclic, quaternaryamine or organic add moieties.
 2. The test device of claim 1 wherein Ris: ##STR8## wherein any of R₁, R₂, R₃, and R₄ are independentlyselected from the group consisting of H, alkyl, aryl, silyl, halide,hydroxide, mercaptide, alkoxide, thioalkoxide, amine, sulfonate orcarboxylate; and X is selected from the group consisting of amine,sulfonate or carboxylate.
 3. The test device of claim 1 wherein saidenzymes are selected from the group consisting of glucose oxidase,cholesterol oxidase, alcohol oxidase, uricase, aldehyde oxidase, andglycerophosphate oxidase.
 4. The test device of claim 3 wherein saidenzymes further comprise peroxidase or inorganic complex havingperoxidase properties.
 5. The test device of claim 3 wherein saidenzymes comprise glucose oxidase and horseradish peroxidase.
 6. The testdevice of claim 1 wherein said dye couple further comprises3-dimethylaminobenzoic acid.
 7. The test device of claim 1 wherein saiddye couple further comprises 8-anilino-1-naphthalenesulfonate.
 8. Thetest device of claim 1 wherein said compound is meta[3-methyl2-benzothiazolinone hydrozone]N-sulfonyl benzenesulfonate monosodium.